T cell-dependent antibody production is a critical mechanism in host defense to various infections. A new CD4+ T cell subset called T follicular helper (Tfh) cells were found to be present in germinal centers and could be identified by their expression of chemokine (C-X-C motif) receptor 5 (CXCR5). In addition to CXCR5, other markers have been also reported for Tfh cells, such as costimulatory receptors ICOS, PD-1 and BTLA, IL-21 cytokine and Bcl-6 transcription factor. Simultaneously with two other groups, Chen Dong group previously showed that Bcl6 serves an indispensable role in T cell-mediated germinal center reactions; mice with Bcl6 deficiency in T cells are impaired in germinal center reactions. More recently, using a new Bcl6 reporter mouse, they found that initial CXCR5 upregulation in T cells precedes that of Bcl6 and is not dependent on Bcl6. Following an immune response, Bcl6+ memory T cells are generated. Hai Qi group has used two-photon intravital microscopy to extensively characterize the localization and movement of Tfh cells in germinal centers. We propose for the current study to understand the genetic factors governing Tfh cell generation and memory differentiation in infectious diseases. The central hypothesis is that distinct transcription factors sequentially promote the initiation, maintenance, and memory formation of the follicle- homing, B cell-interacting, and GC-localizing programs that characterize the Tfh lineage in infectious diseases. We will address this hypothesis under three specific aims. Specific Aim 1, we will understand the mechanism underlying initial CXCR5 upregulation in T cells. In particular, we will study the role of ASCL2 transcription factor, which we recently found to be upregulated in Tfh cells and sufficient in inducing CXCR5 expression. Specific Aim 2, we will study Bcl6 function in Tfh cell development. We will study if Bcl6 is required for proper localization of T cells and their interaction with B cells. In addition, the direct targets of Bcl6will be sought. Specific Aim 3, we will investigate the mechanisms of memory Tfh cell generation. We will analyze the factors required for memory Tfh cell generation and maintenance. Overall, our proposed studies combine the unique expertise from Chen Dong and Hai Qi labs in Tfh cells. By using novel genetic and imaging tools, we are in a unique position in defining the fundamental regulatory mechanisms in Tfh cell generation, function and memory differentiation. The results from our work may help understand humoral immunity against various infections.